Interpretive Summary: Noroviruses (NoV) cause an estimated 23 million annual cases of gastrointestinal disease in the United States, accounting for 50-67% of all food-borne illnesses. A recent CDC survey estimated that 58.3% of NoV infections with a confirmed etiology between 1973 and 2006 were associated with leafy greens. Current methods to recover NoV from large wash volumes from complex samples like leafy greens are ineffective. Histo-blood group antigens (HBGA), which are recognized as a receptor for human NoV, were conjugated to magnetic beads (HBGA-MB) and tested for the ability to recover NoV in food samples. HBGA-conjugated magnetic beads were used and added to 250 ml or larger volumes of wash samples of Romaine lettuce that had been inoculated with a human NoV. NoV were washed off from the surface of lettuce by vigorous shaking, HBGA-MB was added and recovered using a re-circulating immuno-magnetic separation system. NoV recovered by a traditional PEG precipitation method was done also for comparison. NoV was measured by real time RT-PCR. Recovery of NoV by PEG and HBGA-MB was 17% and 42%, respectively. In addition, the HBGA-MB method is 10 times more sensitive than PEG method. Re-circulating immuno-magnetic separation with HBGA-MB is a faster and simpler method than conventional methods for the recovery of NoV from food samples using practical wash volumes.

Technical Abstract:
Noroviruses (NoV) annually cause millions of cases of gastrointestinal disease in the United States. Although NoV outbreaks are generally associated with raw shellfish, particularly oysters, outbreaks have also been known to occur from other common-source food-borne vehicles such as lettuce, frozen raspberries, and salad. In the course of isolating NoV from contaminated produce, a relatively large volume of wash solution has to be used for either immersion or directed rinsing. The virus-containing wash solution is both too voluminous and dilute to be used for detection by RT-PCR without further concentration. While PEG precipitation is the most common method used to concentrate viruses, it is not very sensitive and is time consuming. Recently, histo-blood group antigens (HBGA) have been recognized as ligands for the binding of human norovirus (NoV). Previously, we have demonstrated that HBGA-conjugated magnetic beads (HBGA-MB) can be used to concentrate multiple strains of human NoV effectively from small sample sizes (up to 50 ml) in buffer-diluted samples. However, this technique has never been tested with food samples and the larger liquid volumes that they entail. In this study, we tested the ability and optimal conditions for HBGA-MB recovery of NoV from food samples using a practical 250 ml wash solution volume. NoV recovery was quantitated by real time RT-PCR. The kinetics study suggests 120 min of re-circulation results in best recovery of NoV. Different washing conditions for releasing NoV from inoculated Roman lettuce were compared. NoV released by Citrate buffered saline (CBS), Phosphate buffer saline (PBS) and water are similar, but NoV recovery in CBS is significantly better than PBS. NoV recovery is ineffective when water is used. For comparison, NoV was also recovered by traditional PEG precipitation method. The recovery of NoV from inoculated Romaine lettuce by PEG and PGM-MB methods were 17% and 42%, respectively. When 20, 2, 0.2, 0.02, and 0.002 RT-PCR units (RTU) NoV were spiked in Romaine lettuce wash and concentrated by either PEG or PGM-MB method, 20, 2, 0.2 RTUs could be detected by both methods. For 0.02 RTU, all three samples could be detected by PGM-MB method and two out of three samples could be detected by PEG method. For 0.002 RTU, two out of three samples could be detected by PGM-MB but none could be detected by PGE G method. Recirculation immuno-magnetic separation with PGM-MB is a faster, simpler and more sensitive method than conventional methods for the recovery of NoV from food samples using practical wash volumes.